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D H Turner

Publications and source records attributed to D H Turner.

At least 19 recordsLinked to original sources
PubMed1992

Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods.

Fluorescence-detected stopped-flow and equilibrium methods have been used to study the mechanism for binding of pyrene (pyr)-labeled RNA oligomer substrates to the ribozyme (catalytic RNA) from Tetrahymena thermophila. The fluorescence of these substrates increases up to 25-fold on binding to the ribozyme. Stopped-flow experiments provide evidence that pyr experiences at least three different microenvironments during the binding process. A minimal mechanism is presented in which substrate initially base pairs to ribozyme and subsequently forms tertiary contacts in an RNA folding step. All four microscopic rate constants are measured for ribozyme binding of pyrCCUCU.

Animals↗
PubMed1992

Association of 2'-5' oligoribonucleotides.

Oligoribonucleotides with 2'-5' linkages have been synthesized on solid support. UV melting and CD experiments indicate complementary strands associate to give complexes with melting temperatures 30 to 40 degrees C lower than for duplexes formed by 3'-5' oligoribonucleotides with the same sequence. UV melting and imino proton NMR spectra and NOEs for (2'-5') CGGCGCCG are consistent with formation of an antiparallel duplex. The results suggest greater duplex stability was one factor favoring 3'-5' over 2'-5' linkages in evolution.

Base Sequence↗
PubMed1992

Context dependence of hydrogen bond free energy revealed by substitutions in an RNA hairpin.

Prediction and modeling of RNA structure requires knowledge of the free energy contributions of various interactions. Many unusual hydrogen bonds were recently proposed in the structure of a GCAA hairpin determined from nuclear magnetic resonance. The contributions of these hydrogen bonds to the folding stability of the hairpin formed by rGGCGCAAGCC have now been investigated through the use of functional group substitutions. These and previous results suggest a strong context dependence for the free energy of hydrogen bond formation. The results also suggest that the phylogenetic preference for GNRA (where N = A, C, G, or U and R = A or G) tetraloops may have a functional rather than thermodynamic basis.

Base Sequence↗
PubMed1991

Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text].

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.

Base Composition↗
PubMed1991

Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups.

Dissociation constants at 15 degrees C were measured by equilibrium dialysis for the binding of rCrUrCrU, dCrUrCrU, rCdUrCrU, rCrUdCrU, and rCrUrCdU to the L-21 ScaI form of the self-splicing group I LSU intron from Tetrahymena thermophila. Substitution of deoxyribose for ribose in each of the middle two positions makes the free energy change for binding 1-2 kcal/mol less favorable, compared to about 0.3 kcal/mol less favorable for each of the terminal positions. Dissociation constants for binding of the same oligomers to rGGAGAA were measured by optical melting methods. Substitution of a single deoxyribose for ribose makes the free energy change for binding less favorable by 0.4-0.9 kcal/mol for this simple duplex formation. Comparison of the effects for binding to ribozyme and to rGGAGAA indicate that ribozyme-specific tertiary interactions dependent on the middle two 2' OH groups of rCrUrCrU add about 2 kcal/mol of favorable free energy for binding to L-21 ScaI. Comparisons are made with results from gel retardation studies [Pyle, A. M., McSwiggen, J. A., & Cech, T. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8187-8191; Pyle, A. M., & Cech, T. R. (1991) Nature (London) 350, 628-631].

Animals↗
PubMed1991

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1991

Thermodynamic study of internal loops in oligoribonucleotides: symmetric loops are more stable than asymmetric loops.

Thermodynamic parameters for internal loops of unpaired adenosines in oligoribonucleotides have been measured by optical melting studies. Comparisons are made between helices containing symmetric and asymmetric loops. Asymmetric loops destabilize a helix more than symmetric loops. The differences in free energy between symmetric and asymmetric loops are roughly half the magnitude suggested from a study of parameters required to give accurate predictions of RNA secondary structure [Papanicolaou, C., Gouy, M., & Ninio, J. (1984) Nucleic Acids Res. 12, 31-44]. Circular dichroism spectra indicate no major structural difference between helices containing symmetric and asymmetric loops. The measured sequence dependence of internal loop stability is not consistent with approximations used in current algorithms for predicting RNA secondary structure.

Base Sequence↗
PubMed1991

A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison.

This article describes the latest version of an RNA folding algorithm that predicts both optimal and suboptimal solutions based on free energy minimization. A number of RNA's with known structures deduced from comparative sequence analysis are folded to test program performance. The group of solutions obtained for each molecule is analysed to determine how many of the known helixes occur in the optimal solution and in the best suboptimal solution. In most cases, a structure about 80% correct is found with a free energy within 2% of the predicted lowest free energy structure.

Algorithms↗
PubMed1990

Melting and chemical modification of a cyclized self-splicing group I intron: similarity of structures in 1 M Na+, in 10 mM Mg2+, and in the presence of substrate.

C IVS is the cyclized form of the intron from the RNA precursor of the Tetrahymena thermophila large subunit (LSU) ribosomal RNA. C IVS was mapped by chemical modification in 1 M Na+, 0.05 M Na+ and 10 mM Mg2+ (Na+/Mg2+), and Na+/Mg2+ with CUCU substrate. The results suggest the secondary structure is similar for all three conditions. Optical melting curves were also measured for C IVS in 1 M Na+ and Na+/Mg2+ and indicate the secondary structures have similar stabilities under both conditions. Computer predictions of secondary structure and stability are in good agreement with observations. The results suggest that many of the approximations used for computer prediction of secondary structure by free energy minimization are reasonable.

Aldehydes↗
PubMed1990

Effects of GA mismatches on the structure and thermodynamics of RNA internal loops.

UV melting, CD and NMR studies indicate rGCGAGCG and rGCAGGCG from unusually stable duplexes of type a and b. The observed delta G degree 37's in 1 M NaCl are -6.7 and -6.3 kcal/mol, respectively. For the related duplex, c, delta G degree 37 is -4.2 kcal/mol. The predicted delta G degree 37 from nearest-neighbor parameters (formula; see text) for all three duplexes is -4.7 kcal/mol (Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377). The results suggest a special interaction in the duplexes containing GA mismatches. Presumably, this is hydrogen bonding between G and A. While the thermodynamics for (rGCGAGCG)2 and (rGCAGGCG)2 are similar, CD and the imino region of the proton NMR spectra indicate their structures are different. In particular, (rGCAGGCG)2 exhibits a CD spectrum typical of A-form geometry with a weak negative band at 280 nm. In contrast, the CD spectrum for (rGCGAGCG)2 has an intense positive band at 285 nm. The NMR spectrum of (rGCAGGCG)2 has a resonance corresponding to a hydrogen-bonded GA mismatch, while for (rGCGAGCG)2 no hydrogen-bonded imino proton is observed for the mismatch. The glycosidic torsion angles of the bases in the GA mismatches of (rGCAGGCG)2 and (rCGCAGGCG)2 are anti. Duplexes of type d, where X is A, G, or U, are more stable than e, and the stability differences are similar to those (formula; see text) observed for f versus g. Thus, 3'-dangling ends in this system make contributions to duplex stability that are similar to contributions observed with fully paired duplexes.

Adenosine↗
PubMed1990

Thermodynamic and spectroscopic study of bulge loops in oligoribonucleotides.

Thermodynamic parameters for bulge loops of one to three nucleotides in oligoribonucleotide duplexes have been measured by optical melting. The results indicate bulges Bn of An and Un have similar stabilities in the duplexes, GCGBmGCG + CGCCGC. The stability increment for a bulge depends on more than its adjacent base pairs. For example, the stability increment for a bulge is affected more than 1 kcal/mol by changing two nonadjacent base pairs or by adding terminal unpaired nucleotides (dangling ends) three base pairs away. Thus a nearest-neighbor approximation for helixes with bulges is oversimplified. Many of the non-self-complementary strands used in this study were observed to form homoduplexes. Such duplexes with GA mismatches were particularly stable.

Base Sequence↗
PubMed1989

Improved predictions of secondary structures for RNA.

The accuracy of computer predictions of RNA secondary structure from sequence data and free energy parameters has been increased to roughly 70%. Performance is judged by comparison with structures known from phylogenetic analysis. The algorithm also generates suboptimal structures. On average, the best structure within 10% of the lowest free energy contains roughly 90% of phylogenetically known helixes. The algorithm does not include tertiary interactions or pseudoknots and employs a crude model for single-stranded regions. The only favorable interactions are base pairing and stacking of terminal unpaired nucleotides at the ends of helixes. The excellent performance is consistent with these interactions being the primary interactions determining RNA secondary structure.

Base Composition↗
PubMed1988

Kinetics for reaction of a circularized intervening sequence with CU, UCU, CUCU, and CUCUCU: mechanistic implications from the dependence on temperature and on oligomer and Mg2+ concentrations.

The self-splicing intervening sequence from the rRNA precursor in Tetrahymena thermophila produces a covalently closed, circularized form (C IVS). Reaction rates for reverse cyclization (linearization) of C IVS by the covalent addition of the oligoncleotides CU, UCU, CUCU, and CUCUCU have been measured. The dependence of the observed rates on oligomer and Mg2+ concentrations indicates the presence of intermediates that are generated by separate binding steps for both oligomer and Mg2+. Linearization of C IVS by OH- hydrolysis is suppressed in the presence of oligomer, suggesting oligomer binds near the active site. The binding constants derived for CU at 30 degrees C in 1 and 10 mM Mg2+ are 5 X 10(3) and 2.5 X 10(4) M-1, respectively. These are roughly 4 orders of magnitude larger than expected for simple Watson-Crick base pairing. The binding constants derived for UCU, CUCU, and CUCUCU at 30 degrees C in 10 mM Mg2+ are 1.2 X 10(5), 4 X 10(5), and greater than 10(7) M-1, respectively. The free energy increments for binding of UCU and CUCU relative to CU are similar to those expected from a nearest-neighbor model for addition of base pairs. This indicates the factors responsible for the unusually strong binding of CU to C IVS are restricted to two nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid.

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.

Base Composition↗
PubMed1987

Improved parameters for prediction of RNA structure.

Thermodynamic studies of oligoribonucleotides are providing parameters and insights for the fundamental interactions that determine RNA structure. These results can be used to predict the secondary structure of RNA from its sequence. Comparisons of predicted structures with those deduced from phylogenetic data indicate a modest success rate that is improving as more parameters are determined experimentally. Two major fundamental interactions in RNA are stacking and hydrogen bonding. Both contribute similar increments to free-energy changes for associations of oligoribonucleotides. Thus, parameters for stacking and hydrogen bonding will likely be important for predicting the three-dimensional structures of RNAs and for interpreting RNA-RNA associations. Both applications should be important for providing a full understanding of catalysis by RNA.

Animals↗